CBH1 homologs and variant CBH1 cellulases

ABSTRACT

Disclosed are a number of homologs and variants of  Hypocrea jecorina  Cel7A (formerly  Trichoderma reesei  cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/804,785, filed Mar. 19, 2004, now U.S. Pat. No. 7,452,707 whichclaims priority to U.S. Provisional Application No. 60/456,368 filedMar. 21, 2003 and to U.S. Provisional Application No. 60/458,696 filedMar. 27, 2003, all herein incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Portions of this work were funded by Subcontract No. ZCO-0-30017-01 withthe National Renewable Energy Laboratory under Prime Contract No.DE-AC36-99GO10337 with the U.S. Department of Energy. Accordingly, theUnited States Government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to homologs and variants of Hypocrea jecorina(Trichoderma reesei) CBH1. The present invention relates to isolatednucleic acid sequences which encode polypeptides havingcellobiohydrolase activity. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the nucleic acidsequences as well as methods for producing recombinant variant CBHpolypeptides and novel homologs of H. jecorina CBH1.

REFERENCES

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BACKGROUND OF THE INVENTION

Cellulose and hemicellulose are the most abundant plant materialsproduced by photosynthesis. They can be degraded and used as an energysource by numerous microorganisms, including bacteria, yeast and fungi,that produce extracellular enzymes capable of hydrolysis of thepolymeric substrates to monomeric sugars (Aro et al., 2001). As thelimits of non-renewable resources approach, the potential of celluloseto become a major renewable energy resource is enormous (Krishna et al.,2001). The effective utilization of cellulose through biologicalprocesses is one approach to overcoming the shortage of foods, feeds,and fuels (Ohmiya et al., 1997).

Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or betaD-glucosidic linkages) resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like. Cellulases have beentraditionally divided into three major classes: endoglucanases (EC3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91)(“CBH”) and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC3.2.1.21) (“BG”). (Knowles et al., 1987; Shulein, 1988). Endoglucanasesact mainly on the amorphous parts of the cellulose fibre, whereascellobiohydrolases are also able to degrade crystalline cellulose(Nevalainen and Penttila, 1995). Thus, the presence of acellobiohydrolase in a cellulase system is required for efficientsolubilization of crystalline cellulose (Suumakki, et al. 2000).Beta-glucosidase acts to liberate D-glucose units from cellobiose,cello-oligosaccharides, and other glucosides (Freer, 1993).

Cellulases are known to be produced by a large number of bacteria, yeastand fungi. Certain fungi produce a complete cellulase system capable ofdegrading crystalline forms of cellulose, such that the cellulases arereadily produced in large quantities via fermentation. Filamentous fungiplay a special role since many yeast, such as Saccharomyces cerevisiae,lack the ability to hydrolyze cellulose. See, e.g., Aro et al., 2001;Aubert et al., 1988; Wood et al., 1988, and Coughlan, et al.

The fungal cellulase classifications of CBH, EG and BG can be furtherexpanded to include multiple components within each classification. Forexample, multiple CBHs, EGs and BGs have been isolated from a variety offungal sources including Trichoderma reesei which contains known genesfor 2 CBHs, i.e., CBH1 and CBH II, at least 8 EGs, i.e., EG I, EG II, EGIII, EGIV, EGV, EGVI, EGVII and EGVIII, and at least 5 BGs, i.e., BG1,BG2, BG3, BG4 and BG5.

In order to efficiently convert crystalline cellulose to glucose thecomplete cellulase system comprising components from each of the CBH, EGand BG classifications is required, with isolated components lesseffective in hydrolyzing crystalline cellulose (Filho et al., 1996). Asynergistic relationship has been observed between cellulase componentsfrom different classifications. In particular, the EG-type cellulasesand CBH-type cellulases synergistically interact to more efficientlydegrade cellulose. See, e.g., Wood, 1985.

Cellulases are known in the art to be useful in the treatment oftextiles for the purposes of enhancing the cleaning ability of detergentcompositions, for use as a softening agent, for improving the feel andappearance of cotton fabrics, and the like (Kumar et al., 1997).

Cellulase-containing detergent compositions with improved cleaningperformance (U.S. Pat. No. 4,435,307; GB App. Nos. 2,095,275 and2,094,826) and for use in the treatment of fabric to improve the feeland appearance of the textile (U.S. Pat. Nos. 5,648,263, 5,691,178, and5,776,757; GB App. No. 1,368,599; M. Yamagishi, “Reforming of CellulosicFiber with Cellulase,” The Shizuoka Prefectural Hammamatsu TextileIndustrial Research Institute Report, Vol. 24, pp. 54-61, 1986), havebeen described.

Hence, cellulases produced in fungi and bacteria have receivedsignificant attention. In particular, fermentation of Trichoderma spp.(e.g., Trichoderma longibrachiatum or Trichoderma reesei) has been shownto produce a complete cellulase system capable of degrading crystallineforms of cellulose.

Although cellulase compositions have been previously described, thereremains a need for new and improved cellulase compositions for use inhousehold detergents, stonewashing compositions or laundry detergents,etc. Cellulases that exhibit improved performance are of particularinterest.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated cellulase protein, identified hereinas a desired cellulase, and nucleic acids which encode the desiredcellulase. The desired cellulase may be selected from the groupconsisting of a variant CBH1 from Hypocrea jecorina and a novel CBH1from Hypocrea schweinitzii, Hypocrea odientalis, Trichodermapseudokoningii or Trichoderma konilangbra.

A variant CBH1 cellulase is provided, wherein the variant comprises asubstitution or deletion at a position corresponding to one or more ofresidues L6, S8, P13, Q17, G22, T24, Q27, T41, S47, N49, T59, T66, A68,C71, A77, G88, N89, A100, N103, A112, S113, L125, T160, Y171, Q186,E193, S195, C210, M213, L225, T226, P227, T232, E236, E239, G242, T246,D249, N250, R251, Y252, D257, D259, S278, T281, L288, E295, T296, S297,A299, N301, F311, L318, E325, N327, D329, T332, A336, S341, S342, F352,K354, T356, G359, D368, Y371, N373, T380, Y381, N384, V393, R394, V407,P412, T417, F418 G430, N436, G440, P443, T445, Y466, T478, A481 and/orN490 in CBH1 from Hypocrea jecorina.

In a second aspect, the variant CBH1 comprises a substitution at aposition corresponding to one or more of residues Q186(E), S195(A/F),E239S, G242(H/Y/N/S/T/D/A), D249(K/L/Y/C/I/V/W/T/N/M), E325(S/T),T332(A/H/Y/L/K), and P412(T/S/A).

In a second embodiment the invention provides a Hypocrea orientalisCBH1.

In a third embodiment the invention provides a Hypocrea schweinitziiCBH1.

In a fourth embodiment, there is provided a Trichoderma konilangbraCBH1.

In a fifth embodiment, there is provided a Trichoderma pseudokoningiiCBH1.

In another embodiment of the invention, a nucleic acid that encodes aninventive desired cellulase is provided. In another embodiment, the DNAis in a vector. In a further embodiment, the vector is used to transforma host cell.

In another embodiment of this invention, a method for producing aninventive desired cellulase is provided. The method comprises the stepsof culturing a host cell transformed with a nucleic acid encoding adesired cellulase in a suitable culture medium under suitable conditionsto produce the desired cellulase and obtaining the desired cellulase soproduced.

In yet another embodiment of the invention, a detergent comprising asurfactant and a desired cellulase is provided. In one aspect of thisinvention, the detergent is a laundry or a dish detergent. In secondaspect of this invention, the desired CBH1 cellulase is used in thetreatment of a cellulose containing textile, in particular. In thestonewashing or indigo dyed denim. Alternatively, the cellulase of thisinvention can be used as a feed additive, in the treatment of wood pulp,and in the reduction of biomass to glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid (lower line) (SEQ ID NO:1) and amino acid(upper line) (SEQ ID NO:2) sequence of the wild type Cel7A (CBH1) fromH. jecorina.

FIGS. 2A and 2C show the amino acid alignment of the Cel7A familymembers H. jecorina (also referred to as T. reesei) (SEQ ID NO:2), H.odientalis (SEQ ID NO:5), H. schweinitzii (SEQ ID NO:8), T. konilangbra(SEQ ID NO:11) and T. pseudokoningii (SEQ ID NO:14). The consensussequence is also shown.

FIG. 3 is the genomic DNA sequence for H. orientalis CBH1 (SEQ ID NO:3).Introns are in bold and underlined.

FIG. 4 is the signal sequence (A) (SEQ ID NO:4) and mature amino acidsequence (B) (SEQ ID NO:5) for H. orientalis CBH1.

FIG. 5 is the genomic DNA sequence for H. schweinitzii CBH1 (SEQ IDNO:6). Introns are in bold and underlined.

FIG. 6 is the signal sequence (A) (SEQ ID NO:7) and mature amino acidsequence (B) (SEQ ID NO:8) for H. schweinitzii CBH1.

FIG. 7 is the genomic DNA sequence for T. konilangbra CBH1 (SEQ IDNO:9). Introns are in bold and underlined.

FIG. 8 is the signal sequence (A) (SEQ ID NO:10) and mature amino acidsequence (B) (SEQ ID NO:11) for T. konilangbra CBH1.

FIG. 9 is the genomic DNA sequence for T. pseudokoningii CBH1 (SEQ IDNO:12). Introns are in bold and underlined.

FIG. 10 is the signal sequence (A) (SEQ ID NO:13) and mature amino acidsequence (B) (SEQ ID NO:14) for T. pseudokoningii CBH1.

FIG. 11 is the pRAX1 vector. This vector is based on the plasmid pGAPT2except a 5259 bp HindIII fragment of Aspergillus nidulans genomic DNAfragment AMA1 sequence (Molecular Microbiology 1996 19:565-574) wasinserted. Base 1 to 1134 contains Aspergillus niger glucoamylase genepromoter. Base 3098 to 3356 and 4950 to 4971 contains Aspergillus nigerglucoamylase terminator. Aspergillus nidulans pyrG gene was insertedfrom 3357 to 4949 as a marker for fungal transformation. There is amultiple cloning site (MCS) into which genes may be inserted.

FIG. 12 is the pRAXdes2 vector backbone. This vector is based on theplasmid vector pRAX1. A Gateway cassette has been inserted into pRAX1vector (indicated by the arrow on the interior of the circular plasmid).This cassette contains recombination sequence attR1 and attR2 and theselection marker catH and ccdB. The vector has been made according tothe manual given in Gateway™ Cloning Technology: version 1 page 3438 andcan only replicate in E. coli DB3.1 from Invitrogen; in other E. colihosts the crdB gene is lethal. First a PCR fragment is made with primerscontaining attB1/2 recombination sequences. This fragment is recombinedwith pDONR201 (commercially available from Invitrogen); this vectorcontains attP1/2 recombination sequences with catH and ccdB in betweenthe recombination sites. The BP clonase enzymes from Invitrogen are usedto recombine the PCR fragment in this so-called ENTRY vector, cloneswith the PCR fragment inserted can be selected at 50 μg/ml kanamycinbecause clones expressing ccdB do not survive. Now the aft sequences arealtered and called attL1 and attL2. The second step is to recombine thisclone with the pRAXdes2 vector (containing attR1 and attR2 catH and ccdBin between the recombination sites). The LR clonase enzymes fromInvitrogen are used to recombine the insert from the ENTRY vector in thedestination vector. Only pRAXCBH1 vectors are selected using 100 μg/mlampicillin because ccdB is lethal and the ENTRY vector is sensitive toampicillin. By this method the expression vector is now prepared and canbe used to transform A. niger.

FIG. 13 provides an illustration of the pRAXdes2cbh1 vector which wasused for expression of the nucleic acids encoding the CBH1 homologs orvariants in Aspergillus. A nucleic acid encoding a CBH1 enzyme homologor variant was cloned into the vector by homologous recombination of theatt sequences.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 6 to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

All publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.

I. DEFINITIONS

“Cellulase,” “cellulolytic enzymes” or “cellulase enzymes” meansbacterial, or fungal exoglucanases or exocellobiohydrolases, and/orendoglucanases, and/or β-glucosidases. These three different types ofcellulase enzymes act synergistically to convert cellulose and itsderivatives to glucose.

Many microbes make enzymes that hydrolyze cellulose, including the woodrotting fungus Trichoderma, the compost bacteria Themomonospora,Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola,Aspergillus and Fusarium. The enzymes made by these microbes aremixtures of proteins with three types of actions useful in theconversion of cellulose to glucose: endoglucanases (EG),cellobiohydrolases (CBH), and beta-glucosidase.

A “desired cellulase” as used herein means any one of the following:

-   -   a) a variant CBH1 from Hyprocrea jecorina according to the        present invention;    -   b) a CBH1 homolog from H. orientalis;    -   c) a CBH1 homolog from H. schweinitzii,    -   d) a CBH1 homolog from T. konilangbra;    -   e) a CBH1 homolog from T. pseudokoningii and    -   f) a polypeptide encoded by a nucleic acid that hybridizes with        the nucleic acid that encodes any one of a-e under stringent        conditions.

A “desired cellulase-encoding nucleic acid” as used herein means any oneof the following:

-   -   a) a nucleic acid encoding a variant CBH1 from Hyprocrea        jecorina according to the present invention;    -   b) a nucleic acid encoding a CBH1 homolog from H. orientalis        having the sequence shown in FIG. 3;    -   c) a nucleic acid encoding a CBH1 homolog from H. schweinitzii        having the sequence shown in FIG. 5,    -   d) a nucleic acid encoding a CBH1 homolog from T. konilangbra        having the sequence shown in FIG. 7;    -   e) a nucleic acid encoding a CBH1 homolog from T. pseudokoningii        having the sequence shown in FIG. 9 and    -   f) a nucleic acid that hybridizes with any one of the nucleic        acids provided for by a-e, above, under stringent conditions

“Variant” means a protein which is derived from a precursor protein(e.g., the native protein) by substitution of one or more amino acids atone or a number of different sites in the amino acid sequence. Thepreparation of an enzyme variant is preferably achieved by modifying aDNA sequence which encodes for the native protein, transformation ofthat DNA sequence into a suitable host, and expression of the modifiedDNA sequence to form the derivative enzyme or enzyme variant. Thevariant CBH1 enzyme of the invention includes peptides comprisingaltered amino acid sequences in comparison with a precursor enzyme aminoadd sequence wherein the variant CBH enzyme retains the characteristiccellulolytic nature of the precursor enzyme but which may have alteredproperties in some specific aspect. For example, a variant CBH enzymemay have an increased pH optimum or increased temperature or oxidativestability but will retain its characteristic cellulolytic activity.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, that may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

The “filamentous fungi” of the present invention are eukaryoticmicroorganisms and include all filamentous forms of the subdivisionEumycotina (see Alexopoulos, C. J. (1962), Introductory Mycology, NewYork: Wiley). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi of the present invention aremorphologically, physiologically, and genetically distinct from yeasts.Vegetative growth by filamentous fungi is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as S. cerevisiae is by budding of a unicellular thallus,and carbon catabolism may be fermentative. S. cerevisiae has aprominent, very stable diploid phase, whereas diploids exist onlybriefly prior to melosis in filamentous fungi, e.g., Aspergilli andNeurospora. Although pseudohyphal growth may be exhibited by yeast undercertain conditions it is to be understood that this does not bring theyeast within the definition of filamentous fungi. S. cervisiae has 17chromosomes as opposed to 8 and 7 for A. nidulans and N. crassarespectively. Further illustrations of differences between S. cerevisiaeand filamentous fungi include the inability of S. cerevisiae to processAspergillus and Trichoderma introns and the inability to recognize manytranscriptional regulators of filamentous fungi (Innis, M. A. et al.(1985) Science, 228, 21-26).

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic add comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

A “heterologous” nucleic add construct or sequence has a portion of thesequence which is not native to the cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,transformation, microinjecton, electroporation, or the like. A“heterologous” nucleic acid construct may contain a control sequence/DNAcoding sequence combination that is the same as, or different from acontrol sequence/DNA coding sequence combination found in the nativecell.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As used herein, the terms “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Thepromoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences.

Generally, a “promoter sequence” is a DNA sequence which is recognizedby the particular filamentous fungus for expression purposes. A“constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.An example of an inducible promoter useful in the present invention isthe T. reesei (H. jecorina) cbh1 promoter which is deposited in GenBankunder Accession Number D86235. In another aspect the promoter is a cbhII or xylanase promoter from H. jecorina.

Exemplary promoters include the promoter from the A. awamori or A. nigerglucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell. Biol. 4,2306-2315; Boel, E. et al. (1984) EMBO J. 3, 1581-1585), the Mucormiehei carboxyl protease gene, the Hypocrea jecorina cellobiohydrolase Igene (Shoemaker, S. P. et al. (1984) European Patent Application No.EP00137280A1), the A. nidulans trpC gene (Yelton, M. et al. (1984) Proc.Natl. Acad. Sci. USA 81, 1470-1474; Mullaney, E. J. et al. (1985) Mol.Gen. Genet. 199, 37-45) the A. nidulans alcA gene (Lockington, R. A. etal. (1985) Gene 33, 137-149), the A. nidulans tpiA gene (McKnight, G. L.et al. (1986) Cell 46, 143-147), the A. nidulans amdS gene (Hynes, M. J.et al. (1983) Mol. Cell Biol. 3, 1430-1439), the H. jecorina xln1 gene,the H. jecorina cbh2 gene, the H. jecorina eg1 gene, the H. jecorina eg2gene, the H. jecorina eg3 gene, and higher eukaryotic promoters such asthe SV40 early promoter (Barclay, S. L. and E. Meller (1983) Molecularand Cellular Biology 3, 2117-2130).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader, i.e., a signal peptide, is operably linkedto DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice. Thus, theterm “operably linked” refers to a functional linkage between a nucleicadd expression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

“Chimeric gene” or “heterologous nucleic acid construct”, as definedherein refers to a non-native gene (i.e., one that has been introducedinto a host) that may be composed of parts of different genes, includingregulatory elements. A chimeric gene construct for transformation of ahost cell is typically composed of a transcriptional regulatory region(promoter) operably linked to a heterologous protein coding sequence,or, in a selectable marker chimeric gene, to a selectable marker geneencoding a protein conferring antibiotic resistance to transformedcells. A typical chimeric gene of the present invention, fortransformation into a host cell, includes a transcriptional regulatoryregion that is constitutive or inducible, a protein coding sequence, anda terminator sequence. A chimeric gene construct may also include asecond DNA sequence encoding a signal peptide if secretion of the targetprotein is desired.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

As used herein, the phrases “whole cellulase preparation” and “wholecellulase composition” are used interchangeably and refer to bothnaturally occurring and non-naturally occurring compositions. A“naturally occurring” composition is one produced by a naturallyoccurring source and which comprises one or more cellobiohydrolase-type,one or more endoglucanase-type, and one or more β-glucosidase componentswherein each of these components is found at the ratio produced by thesource. A naturally occurring composition is one that is produced by anorganism unmodified with respect to the cellulolytic enzymes such thatthe ratio of the component enzymes is unaltered from that produced bythe native organism.

A “non-naturally occurring” composition encompasses those compositionsproduced by: (1) combining component cellulolytic enzymes either in anaturally occurring ratio or non-naturally occurring, i.e., altered,ratio; or (2) modifying an organism to overexpress or underexpress oneor more cellulolytic enzyme; or (3) modifying an organism such that atleast one cellulolytic enzyme is deleted.

“Equivalent residues” may also be defined by determining homology at thelevel of tertiary structure for a precursor cellulase whose tertiarystructure has been determined by x-ray crystallography. Equivalentresidues are defined as those for which the atomic coordinates of two ormore of the main chain atoms of a particular amino acid residue of acellulase and Hypocrea jecorina CBH (N on N, CA on CA, C on C and O onO) are within 0.13 nm and preferably 0.1 nm after alignment. Alignmentis achieved after the best model has been oriented and positioned togive the maximum overlap of atomic coordinates of non-hydrogen proteinatoms of the cellulase in question to the H. jecorina CBH1. The bestmodel is the crystallographic model giving the lowest R factor forexperimental diffraction data at the highest resolution available.

${Rfactor} = \frac{{\Sigma_{h}{{{Fo}(h)}}} - {{{Fc}(h)}}}{\Sigma_{h}{{{Fo}(h)}}}$

Equivalent residues which are functionally analogous to a specificresidue of H. jecorina CBH 1 are defined as those amino adds of acellulase which may adopt a conformation such that they either alter,modify or contribute to protein structure, substrate binding orcatalysis in a manner defined and attributed to a specific residue ofthe H. jecorina CBH1. Further, they are those residues of the cellulase(for which a tertiary structure has been obtained by x-raycrystallography) which occupy an analogous position to the extent that,although the main chain atoms of the given residue may not satisfy thecriteria of equivalence on the basis of occupying a homologous position,the atomic coordinates of at least two of the side chain atoms of theresidue lie with 0.13 nm of the corresponding side chain atoms of H.jecorina CBH.

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules.It will be understood that, as a result of the degeneracy of the geneticcode, a multitude of nucleotide sequences encoding a given protein suchas CBH1 may be produced. The present invention contemplates everypossible variant nucleotide sequence, encoding CBH1, all of which arepossible given the degeneracy of the genetic code.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

Accordingly, an “expression cassette” or “expression vector” is anucleic acid construct generated recombinantly or synthetically, with aseries of specified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in many bacteria andsome eukaryotes.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence which is capable of expressionin cells and where expression of the selectable marker confers to cellscontaining the expressed gene the ability to grow in the presence of acorresponding selective agent, or under corresponding selective growthconditions.

In general, nucleic acid molecules which encode the variant CBH1 willhybridize, under moderate to high stringency conditions to the wild typesequence provided herein as SEQ ID NO: 1 (native H. jecorina CBH1).However, in some cases a CBH1-encoding nucleotide sequence is employedthat possesses a substantially different codon usage, while the proteinencoded by the CBH1-encoding nucleotide sequence has the same orsubstantially the same amino acid sequence as the native protein. Forexample, the coding sequence may be modified to facilitate fasterexpression of CBH1 in a particular prokaryotic or eukaryotic expressionsystem, in accordance with the frequency with which a particular codonis utilized by the host. Te'o, et al. (2000), for example, describes theoptimization of genes for expression in filamentous fungi.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “moderate” or “intermediate stringency” at about 10-20° below the Tmof the probe; and “low stringency” at about 20-25° below the Tm.Functionally, maximum stringency conditions may be used to Identifysequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify sequences having about 80% or more sequence identity with theprobe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, andin Ausubel, F. M., et al., 1993, expressly incorporated by referenceherein). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDSand 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome or as anepisomal plasmid that is maintained through multiple generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

It follows that the term “desired cellulase expression” refers totranscription and translation of the desired cellulase gene, theproducts of which include precursor RNA, mRNA, polypeptide,post-translationally processed polypeptides. By way of example, assaysfor CBH1 expression include Western blot for CBH1 protein, Northern blotanalysis and reverse transcriptase polymerase chain reaction (RT-PCR)assays for CBH1 mRNA, and endoglucanase activity assays as described inShoemaker S. P. and Brown R. D. Jr. (Biochim. Biophys. Acta, 1978,523:133-146) and Schulein (1988).

The term “alternative splicing” refers to the process whereby multiplepolypeptide isoforms are generated from a single gene, and involves thesplicing together of nonconsecutive exons during the processing of some,but not all, transcripts of the gene. Thus a particular exon may beconnected to any one of several alternative exons to form messengerRNAs. The alternatively-spliced mRNAs produce polypeptides (“splicevariants”) in which some parts are common while other parts aredifferent.

By the term “host-cell” is meant a cell that contains a vector andsupports the replication, and/or transcription or transcription andtranslation (expression) of the expression construct. Host cells for usein the present invention can be prokaryotic cells, such as E. coli, oreukaryotic cells such as yeast, plant, insect, amphibian, or mammaliancells. In general, host cells are filamentous fungi.

The term “cellulase” refers to a category of enzymes capable ofhydrolyzing cellulose polymers to shorter cello-oligosaccharideoligomers, cellobiose and/or glucose. Numerous examples of cellulases,such as exoglucanases, exocellobiohydrolases, endoglucanases, andglucosidases have been obtained from cellulolytic organisms,particularly including fungi, plants and bacteria.

CBH1 from Hypocrea jecorina is a member of the Glycosyl Hydrolase Family7 (hence Cel7) and, specifically, was the first member of that familyidentified in Hypocrea jecorina (hence Cel7A). The Glycosyl HydrolaseFamily 7 contains both Endoglucanases andCellobiohydrolases/exoglucanases, and that CBH1 is the latter. Thus, thephrases CBH1, CBH1-type protein and Cel7 cellobiohydrolases may be usedinterchangeably herein.

The term “cellulose binding domain” as used herein refers to portion ofthe amino acid sequence of a cellulase or a region of the enzyme that isinvolved in the cellulose binding activity of a cellulase or derivativethereof. Cellulose binding domains or modules generally function bynon-covalently binding the cellulase to cellulose, a cellulosederivative or other polysaccharide equivalent thereof. Cellulose bindingdomains permit or facilitate hydrolysis of cellulose fibers by thestructurally distinct catalytic core region, and typically functionindependent of the catalytic core. Thus, a cellulose binding domain willnot possess the significant hydrolytic activity attributable to acatalytic core. In other words, a cellulose binding domain is astructural element of the cellulase enzyme protein tertiary structurethat is distinct from the structural element which possesses catalyticactivity.

As used herein, the term “surfactant” refers to any compound generallyrecognized in the art as having surface active qualities. Thus, forexample, surfactants comprise anionic, cationic and nonionic surfactantssuch as those commonly found in detergents. Anionic surfactants includelinear or branched alkylbenzenesulfonates; alkyl or alkenyl ethersulfates having linear or branched alkyl groups or alkenyl groups; alkylor alkenyl sulfates; olefinsulfonates; and alkanesulfonates. Ampholyticsurfactants include quaternary ammonium salt sulfonates, andbetaine-type ampholytic surfactants. Such ampholytic surfactants haveboth the positive and negative charged groups in the same molecule.Nonionic surfactants may comprise polyoxyalkylene ethers, as well ashigher fatty acid alkanolamides or alkylene oxide adduct thereof, fattyadd glycerine monoesters, and the like.

As used herein, the term “cellulose containing fabric” refers to anysewn or unsewn fabrics, yarns or fibers made of cotton or non-cottoncontaining cellulose or cotton or non-cotton containing cellulose blendsincluding natural cellulosics and manmade cellulosics (such as jute,flax, ramie, rayon, and lyocell).

As used herein, the term “cotton-containing fabric” refers to sewn orunsewn fabrics, yarns or fibers made of pure cotton or cotton blendsincluding cotton woven fabrics, cotton knits, cotton denims, cottonyarns, raw cotton and the like.

As used herein, the term “stonewashing composition” refers to aformulation for use in stonewashing cellulose containing fabrics.Stonewashing compositions are used to modify cellulose containingfabrics prior to sale, i.e., during the manufacturing process. Incontrast, detergent compositions are intended for the cleaning of soiledgarments and are not used during the manufacturing process.

As used herein, the term “detergent composition” refers to a mixturewhich is intended for use in a wash medium for the laundering of soiledcellulose containing fabrics. In the context of the present invention,such compositions may include, in addition to cellulases andsurfactants, additional hydrolytic enzymes, builders, bleaching agents,bleach activators, bluing agents and fluorescent dyes, cakinginhibitors, masking agents, cellulase activators, antioxidants, andsolubilizers.

As used herein, the term “decrease or elimination in expression of thecbh1 gene” means that either that the cbh1 gene has been deleted fromthe genome and therefore cannot be expressed by the recombinant hostmicroorganism; or that the cbh1 gene has been modified such that afunctional CBH1 enzyme is not produced by the host microorganism.

The term “variant cbh1 gene” or “variant CBH1” means, respectively, thatthe nucleic acid sequence of the cbh1 gene from H. jecorina has beenaltered by removing, adding, and/or manipulating the coding sequence orthe amino acid sequence of the expressed protein has been modifiedconsistent with the invention described herein.

As used herein, the terms “active” and “biologically active” refer to abiological activity associated with a particular protein and are usedinterchangeably herein. For example, the enzymatic activity associatedwith a protease is proteolysis and, thus, an active protease hasproteolytic activity. It follows that the biological activity of a givenprotein refers to any biological activity typically attributed to thatprotein by those of skill in the art.

When employed in enzymatic solutions, the homolog or variant CBH1component is generally added in an amount sufficient to allow thehighest rate of release of soluble sugars from the biomass. The amountof homolog or variant CBH1 component added depends upon the type ofbiomass to be saccharified which can be readily determined by theskilled artisan. However, when employed, the weight percent of thehomolog or variant CBH1 component relative to any EG type componentspresent in the cellulase composition is from preferably about 1,preferably about 5, preferably about 10, preferably about 15, orpreferably about 20 weight percent to preferably about 25, preferablyabout 30, preferably about 35, preferably about 40, preferably about 45or preferably about 50 weight percent. Furthermore, preferred ranges maybe about 0.5 to about 15 weight percent, about 0.5 to about 20 weightpercent, from about 1 to about 10 weight percent, from about 1 to about15 weight percent, from about 1 to about 20 weight percent, from about 1to about 25 weight percent, from about 5 to about 20 weight percent,from about 5 to about 25 weight percent, from about 5 to about 30 weightpercent, from about 5 to about 35 weight percent, from about 5 to about40 weight percent, from about 5 to about 45 weight percent, from about 5to about 50 weight percent, from about 10 to about 20 weight percent,from about 10 to about 25 weight percent, from about 10 to about 30weight percent, from about 10 to about 35 weight percent, from about 10to about 40 weight percent, from about 10 to about 45 weight percent,from about 10 to about 50 weight percent, from about 15 to about 20weight percent, from about 15 to about 25 weight percent, from about 15to about 30 weight percent, from about 15 to about 35 weight percent,from about 15 to about 30 weight percent, from about 15 to about 45weight percent, from about 15 to about 50 weight percent.

II. HOST ORGANISMS

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota. The filamentous fungi are characterized byvegetative mycelium having a cell wall composed of chitin, glucan,chitosan, mannan, and other complex polysaccharides, with vegetativegrowth by hyphal elongation and carbon catabolism that is obligatelyaerobic.

In the present invention, the filamentous fungal parent cell may be acell of a species of, but not limited to, Trichoderma, e.g., Trichodermalongibrachiatum, Trichoderma viride, Trichoderma koningii, Trichodermaharianum; Penicillium sp.; Humicola sp., including Humicola insolens andHumicola grisea; Chrysosporium sp., including C. lucknowense;Gliocladium sp.; Aspergillus sp.; Fusatium sp., Neurospora sp., Hypocreasp., and Emericella sp. As used herein, the term “Trichoderma” or“Trichoderma sp.” refers to any fungal strains which have previouslybeen classified as Trichoderma or are currently classified asTrichoderma.

In one preferred embodiment, the filamentous fungal parent cell is anAspergillus niger, Aspergillus awamori, Aspergillus aculeatus, orAspergillus nidulans cell.

In another preferred embodiment, the filamentous fungal parent cell is aTrichoderma reesei cell.

III. CELLULASES

Cellulases are known in the art as enzymes that hydrolyze cellulose(beta-1,4-glucan or beta D-glucosidic linkages) resulting in theformation of glucose, cellobiose, cellooligosaccharides, and the like.As set forth above, cellulases have been traditionally divided intothree major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanasesor cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases (EC3.2.1.21) (“BG”). (Knowles, et al., 1987; Schulein, 1988).

Certain fungi produce complete cellulase systems which includeexo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-typecellulases and beta-glucosidases or BG-type cellulases (Schulein, 1988).However, sometimes these systems lack CBH-type cellulases and bacterialcellulases also typically include little or no CBH-type cellulases. Inaddition, it has been shown that the EG components and CBH componentssynergistically interact to more efficiently degrade cellulose. See,e.g., Wood, 1985. The different components, i.e., the variousendoglucanases and exocellobiohydrolases in a multi-component orcomplete cellulase system, generally have different properties, such asisoelectric point, molecular weight, degree of glycosylation, substratespecificity and enzymatic action patterns.

Cellulase compositions have also been shown to degrade cotton-containingfabrics, resulting in reduced strength loss in the fabric (U.S. Pat. No.4,822,516), contributing to reluctance to use cellulase compositions incommercial detergent applications. Cellulase compositions comprisingendoglucanase components have been suggested to exhibit reduced strengthloss for cotton-containing fabrics as compared to compositionscomprising a complete cellulase system.

Cellulases have also been shown to be useful in degradation of cellulasebiomass to ethanol (wherein the cellulase degrades cellulose to glucoseand yeast or other microbes further ferment the glucose into ethanol),in the treatment of mechanical pulp (Pere et al., 1996), for use as afeed additive (WO 91/04673) and in grain wet milling.

Most CBHs and EGs have a multidomain structure consisting of a coredomain separated from a cellulose binding domain (CBD) by a linkerpeptide (Suumakki et al., 2000). The core domain contains the activesite whereas the CBD interacts with cellulose by binding the enzyme toit (van Tilbeurgh et al., 1986; Tomme et al., 1988). The CBDs areparticularly important in the hydrolysis of crystalline cellulose. Ithas been shown that the ability of cellobiohydrolases to degradecrystalline cellulose clearly decreases when the CBD is absent (Linderand Teeri, 1997). However, the exact role and action mechanism of CBDsis still a matter of speculation. It has been suggested that the CBDenhances the enzymatic activity merely by increasing the effectiveenzyme concentration at the surface of cellulose (Stahlberg et al.,1991), and/or by loosening single cellulose chains from the cellulosesurface (Tormo et al., 1996). Most studies concerning the effects ofcellulase domains on different substrates have been carried out withcore proteins of cellobiohydrolases, as their core proteins can easilybe produced by limited proteolysis with papain (Tomme et al., 1988).Numerous cellulases have been described in the scientific literature,examples of which include: from Trichoderma reesei Shoemaker, S. et al.,Bio/Technology, 1:691-696, 1983, which discloses CBHI; Teeri, T. et al.,Gene, 51:43-52, 1987, which discloses CBHII. Cellulases from speciesother than Trichoderma have also been described e.g., Ooi et al., 1990,which discloses the cDNA sequence coding for endoglucanase F1-CMCproduced by Aspergillus aculeatus; Kawaguchi T et al., 1996, whichdiscloses the cloning and sequencing of the cDNA encodingbeta-glucosidase 1 from Aspergillus aculeatus; Sakamoto et al., 1995,which discloses the cDNA sequence encoding the endoglucanase CMCase-1from Aspergillus kawachii IFO 4308; Saarilahti et al, 1990 whichdiscloses an endoglucanase from Erwinia carotovara; Spilliaert R, etal., 1994, which discloses the cloning and sequencing of bglA, codingfor a thermostable beta-glucanase from Rhodothermus marinus; andHaldorsdottir S et al., 1998, which discloses the cloning, sequencingand overexpression of a Rhodothermus marinus gene encoding athermostable cellulase of glycosyl hydrolase family 12. However, thereremains a need for identification and characterization of novelcellulases, with improved properties, such as improved performance underconditions of thermal stress or in the presence of surfactants,increased specific activity, altered substrate cleavage pattern, and/orhigh level expression in vitro.

The development of new and improved cellulase compositions that comprisevarying amounts CBH-type cellulase is of interest for use: (1) indetergent compositions that exhibit enhanced cleaning ability, functionas a softening agent and/or improve the feel of cotton fabrics (e.g.,“stone washing” or “biopolishing”); (2) in compositions for degradingwood pulp or other biomass into sugars (e.g., for bio-ethanolproduction); and/or (3) in feed compositions.

IV. MOLECULAR BIOLOGY

In one embodiment this invention provides for the expression of desiredcellulase genes under control of a promoter functional in a filamentousfungus. Therefore, this invention relies on routine techniques in thefield of recombinant genetics. Basic texts disclosing the generalmethods of use in this invention include Sambrook et al., MolecularCloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Ausubel et al., eds.,Current Protocols in Molecular Biology (1994)).

A. Methods for Identifying Homologous CBH1 Genes

The nucleic acid sequence for the wild type H. jecorina CBH1 is shown inFIG. 1. The invention, in one aspect, encompasses a nucleic acidmolecule encoding a CBH I homolog described herein. The nucleic acid maybe a DNA molecule.

Techniques that can be used to isolate homologous CBH1-encoding DNAsequences are well known in the art and include, but are not limited to,cDNA and/or genomic library screening with a homologous DNA probes andexpression screening with activity assays or antibodies against CBH1.Any of these methods can be found in Sambrook, et al. or in CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel, et al., ed. GreenePublishing and Wiley-Interscience, New York (1987) (“Ausubel”).

B. Methods of Mutating CBH Nucleic Acid Sequences

Any method known in the art that can introduce mutations is contemplatedby the present invention.

The present invention relates to the expression, purification and/orisolation and use of variant CBH1. These enzymes are preferably preparedby recombinant methods utilizing the cbh gene from H. jecorina.

After the isolation and cloning of the cbh1 gene from H. jecorina, othermethods known in the art, such as site directed mutagenesis, are used tomake the substitutions, additions or deletions that correspond tosubstituted amino acids in the expressed CBH1 variant. Again, sitedirected mutagenesis and other methods of incorporating amino acidchanges in expressed proteins at the DNA level can be found in Sambrook,et al. and Ausubel, et al.

DNA encoding an amino acid sequence variant of the H. jecorina CBH1 isprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the H. jecorina CBH1.

Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (see,e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel etal., Proc. Natl. Acad. Sci. USA 82:488 (1985) (1987)). Briefly, incarrying out site-directed mutagenesis of DNA, the starting DNA isaltered by first hybridizing an oligonucleotide encoding the desiredmutation to a single strand of such starting DNA. After hybridization, aDNA polymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide, i.e., H. jecorina CBH1. See Higuchi, in PCRProtocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc.Acids Res. 17:723-733 (1989). Briefly, when small amounts of templateDNA are used as starting material in a PCR, primers that differ slightlyin sequence from the corresponding region in a template DNA can be usedto generate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene 34:315-323 (1985). Thestarting material is the plasmid (or other vector) comprising thestarting polypeptide DNA to be mutated. The codon(s) in the starting DNAto be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the starting polypeptide DNA. Theplasmid DNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence.

Alternatively, or additionally, the desired amino acid sequence encodinga desired cellulase can be determined, and a nucleic acid sequenceencoding such amino acid sequence variant can be generatedsynthetically.

The desired cellulase(s) so prepared may be subjected to furthermodifications, oftentimes depending on the intended use of thecellulase. Such modifications may involve further alteration of theamino acid sequence, fusion to heterologous polypeptide(s) and/orcovalent modifications.

V. cbh1 NUCLEIC ACIDS AND CBH1 POLYPEPTIDES A. Variant cbh-Type NucleicAcids

After DNA sequences that encode the CBH1 variants have been cloned intoDNA constructs, the DNA is used to transform microorganisms. Themicroorganism to be transformed for the purpose of expressing a variantCBH1 according to the present invention may advantageously comprise astrain derived from Trichoderma sp. Thus, a preferred mode for preparingvariant CBH1 cellulases according to the present invention comprisestransforming a Trichoderma sp. host cell with a DNA construct comprisingat least a fragment of DNA encoding a portion or all of the variantCBH1. The DNA construct will generally be functionally attached to apromoter. The transformed host cell is then grown under conditions so asto express the desired protein. Subsequently, the desired proteinproduct is purified to substantial homogeneity.

However, it may in fact be that the best expression vehicle for a givenDNA encoding a variant CBH1 may differ from H. jecorina. Thus, it may bethat it will be most advantageous to express a protein in atransformation host that bears phylogenetic similarity to the sourceorganism for the variant CBH1. In an alternative embodiment, Aspergillusniger can be used as an expression vehicle. For a description oftransformation techniques with A. niger, see WO 98/31821, the disclosureof which is incorporated by reference in its entirety.

Accordingly, the present description of a Trichoderma spp. expressionsystem is provided for illustrative purposes only and as one option forexpressing the variant CBH1 of the invention. One of skill in the art,however, may be inclined to express the DNA encoding variant CBH1 in adifferent host cell if appropriate and it should be understood that thesource of the variant CBH1 should be considered in determining theoptimal expression host. Additionally, the skilled worker in the fieldwill be capable of selecting the best expression system for a particulargene through routine techniques utilizing the tools available in theart.

B. Variant CBH1 Polypeptides

The amino acid sequence for the wild type H. jecorina CBH1 is shown inFIG. 1. The variant CBH1 polypeptides comprise a substitution ordeletion at a position corresponding to one or more of residues L6, S8,P13, Q17, G22, T24, Q27, T41, S47, N49, T59, T66, A68, C71, A77, G88,N89, A100, N103, A112, S113, L125, T160, Y171, Q186, E193, S195, C210,M213, L225, T226, P227, T232, E236, E239, G242, T246, D249, N250, R251,Y252, D257, D259, S278, T281, L288, E295, T296, S297, A299, N301, F311,L318, E325, N327, D329, T332, A336, S341, S342, F352, K354, T356, G359,D368, Y371, N373, T380, Y381, N384, V393, R394, V407, P412, T417, F418G430, N436, G440, P443, T445, Y466, T478, A481 and/or N490 in CBH1 fromHypocrea jecorina.

The variant CBH1's of this invention have amino acid sequences that arederived from the amino acid sequence of a precursor H. jecorina CBH1.The amino acid sequence of the CBH1 variant differs from the precursorCBH1 amino acid sequence by the substitution, deletion or insertion ofone or more amino acids of the precursor amino acid sequence. The matureamino acid sequence of H. jecorina CBH1 is shown in FIG. 1. Thus, thisinvention is directed to CBH1 variants which contain amino acid residuesat positions which are equivalent to the particular identified residuein H. jecorina CBH1. A residue (amino acid) of an CBH1 variant isequivalent to a residue of Hypocrea jecorina CBH1 if it is eitherhomologous (i.e., corresponding in position in either primary ortertiary structure) or is functionally analogous to a specific residueor portion of that residue in Hypocrea jecorina CBH1 (i.e., having thesame or similar functional capacity to combine, react, or interactchemically or structurally). As used herein, numbering is intended tocorrespond to that of the mature CBH1 amino acid sequence as illustratedin FIG. 1. In addition to locations within the precursor CBH1, specificresidues in the precursor CBH1 corresponding to the amino acid positionsthat are responsible for instability when the precursor CBH1 is underthermal stress are identified herein for substitution or deletion. Theamino acid position number (e.g., +51) refers to the number assigned tothe mature Hypocrea jecorina CBH1 sequence presented in FIG. 1.

Alignment of amino acid sequences to determine homology is preferablydetermined by using a “sequence comparison algorithm.” Optimal alignmentof sequences for comparison can be conducted, e.g., by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), by visual inspection or MOE by ChemicalComputing Group, Montreal Canada.

An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol. 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. These initial neighborhood word hits actas starting points to find longer HSPs containing them. The word hitsare expanded in both directions along each of the two sequences beingcompared for as far as the cumulative alignment score can be increased.Extension of the word hits is stopped when: the cumulative alignmentscore falls off by the quantity X from a maximum achieved value; thecumulative score goes to zero or below; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, (1992))alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (see, e.g., Kadin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, an amino acid sequence is considered similar to a protease ifthe smallest sum probability in a comparison of the test amino acidsequence to a protease amino acid sequence is less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.001.

Additional specific strategies for modifying stability of CBH1cellulases are provided below:

(1) Decreasing the entropy of main-chain unfolding may introducestability to the enzyme. For example, the introduction of prolineresidues may significantly stabilize the protein by decreasing theentropy of the unfolding (see, e.g., Watanabe, et al., Eur. J. Biochem.226:277-283 (1994)). Similarly, glycine residues have no β-carbon, andthus have considerably greater backbone conformational freedom than manyother residues. Replacement of glycines, preferably with alanines, mayreduce the entropy of unfolding and improve stability (see, e.g.,Matthews, et al., Proc. Natl. Acad. Sci. USA 84; 6663-6667 (1987)).Additionally, by shortening external loops it may be possible to improvestability. It has been observed that hyperthermophile produced proteinshave shorter external loops than their mesophilic homologues (see, e.g.,Russell, et al., Current Opinions in Biotechnology 6:370-374 (1995)).The introduction of disulfide bonds may also be effective to stabilizedistinct tertiary structures in relation to each other. Thus, theintroduction of cysteines at residues accessible to existing cysteinesor the introduction of pairs of cysteines that could form disulfidebonds would alter the stability of a CBH1 variant.

(2) Decreasing internal cavities by increasing side-chain hydrophobicitymay alter the stability of an enzyme. Reducing the number and volume ofinternal cavities increases the stability of enzyme by maximizinghydrophobic interactions and reducing packing defects (see, e.g.,Matthews, Ann. Rev. Biochem. 62:139-160 (1993); Burley, et al., Science229:23-29 (1985); Zuber, Biophys. Chem. 29:171-179 (1988); Kellis, etal., Nature 333:784-786 (1988)). It is known that multimeric proteinsfrom thermophiles often have more hydrophobic sub-unit interfaces withgreater surface complementarity than their mesophilic counterparts(Russell, et al., supra). This principle is believed to be applicable todomain interfaces of monomeric proteins. Specific substitutions that mayimprove stability by increasing hydrophobicity include lysine toarginine, serine to alanine and threonine to alanine (Russell, et al.,supra). Modification by substitution to alanine or proline may increaseside-chain size with resultant reduction in cavities, better packing andincreased hydrophobicity. Substitutions to reduce the size of thecavity, increase hydrophobicity and improve the complementarity theinterfaces between the domains of CBH1 may improve stability of theenzyme. Specifically, modification of the specific residue at thesepositions with a different residue selected from any of phenylalanine,tryptophan, tyrosine, leucine and isoleucine may improve performance.

(3) Balancing charge in rigid secondary structure, i.e., α-helices andβ-turns may improve stability. For example, neutralizing partialpositive charges on a helix N-terminus with negative charge on asparticacid may improve stability of the structure (see, e.g., Eriksson, etal., Science 255:178-183 (1992)). Similarly, neutralizing partialnegative charges on helix C-terminus with positive charge may improvestability. Removing positive charge from interacting with peptideN-terminus in β-turns should be effective in conferring tertiarystructure stability. Substitution with a non-positively charged residuecould remove an unfavorable positive charge from interacting with anamide nitrogen present in a turn.

(4) Introducing salt bridges and hydrogen bonds to stabilize tertiarystructures may be effective. For example, ion pair interactions, e.g.,between aspartic acid or glutamic acid and lysine, arginine orhistidine, may introduce strong stabilizing effects and may be used toattach different tertiary structure elements with a resultantimprovement in thermostability. Additionally, increases in the number ofcharged residue/non-charged residue hydrogen bonds, and the number ofhydrogen-bonds generally, may improve thermostability (see, e.g.,Tanner, et al., Biochemistry 35:2597-2609 (1996)). Substitution withaspartic acid, asparagine, glutamic acid or glutamine may introduce ahydrogen bond with a backbone amide. Substitution with arginine mayimprove a salt bridge and introduce an H-bond into a backbone carbonyl.

(5) Avoiding thermolabile residues in general may increase thermalstability. For example, asparagine and glutamine are susceptible todeamidation and cysteine is susceptible to oxidation at hightemperatures. Reducing the number of these residues in sensitivepositions may result in improved thermostability (Russell, et al.,supra). Substitution or deletion by any residue other than glutamine orcysteine may increase stability by avoidance of a thermolabile residue.

(6) Stabilization or destabilization of binding of a ligand that confersmodified stability to CBH1 variants. For example, a component of thematrix in which the CBH1 variants of this invention are used may bind toa specific surfactant/thermal sensitivity site of the CBH1 variant. Bymodifying the site through substitution, binding of the component to thevariant may be strengthened or diminished. For example, a non-aromaticresidue in the binding crevice of CBH1 may be substituted withphenylalanine or tyrosine to introduce aromatic side-chain stabilizationwhere interaction of the cellulose substrate may interact favorably withthe benzyl rings, increasing the stability of the CBH1 variant.

(7) Increasing the electronegatvity of any of the surfactant thermalsensitivity ligands may improve stability under surfactant or thermalstress. For example, substitution with phenylalanine or tyrosine mayincrease the electronegativity of D (aspartate) residues by improvingshielding from solvent, thereby improving stability.

C. Homologous CBH1 Nucleic Acids and Polypeptides

Genomic DNA from microbial organisms is fixed to a membrane. The genomicDNA is hybridized with the gene specific probes and screened using PCR.The PCR product(s) are isolated using techniques well known in the artand sequenced.

VI. EXPRESSION OF RECOMBINANT CBH1 HOMOLOGS AND VARIANTS

The methods of the invention rely on the use cells to express a desiredcellulase, with no particular method of expression required.

The invention provides host cells that have been transduced, transformedor transfected with an expression vector comprising a desiredcellulase-encoding nucleic acid sequence. The culture conditions, suchas temperature, pH and the like, are those previously used for theparental host cell prior to transduction, transformation or transfectionand will be apparent to those skilled in the art.

In one approach, a filamentous fungal cell or yeast cell is transfectedwith an expression vector having a promoter or biologically activepromoter fragment or one or more (e.g., a series) of enhancers whichfunctions in the host cell line, operably linked to a DNA segmentencoding a desired cellulase, such that desired cellulase is expressedin the cell line.

A. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding a desiredcellulase (“desired cellulase-encoding nucleic acid sequences”) may beincorporated into heterologous nucleic acid constructs or vectors,capable of introduction into, and replication in, a filamentous fungalor yeast cell. The vectors and methods disclosed herein are suitable foruse in host cells for the expression of a desired cellulase. Any vectormay be used as long as it is replicable and viable in the cells intowhich it is introduced. Large numbers of suitable vectors and promotersare known to those of skill in the art, and are commercially available.Cloning and expression vectors are also described in Sambrook et al.,1989, Ausubel F M et al., 1989, and Strathern et al., 1981, each ofwhich is expressly incorporated by reference herein. Appropriateexpression vectors for fungi are described in van den Hondel, C. A. M.J. J. et al. (1991) In: Bennett, J. W. and Lasure, L. L. (eds.) MoreGene Manipulations in Fungi. Academic Press, pp. 396428. The appropriateDNA sequence may be inserted into a plasmid or vector (collectivelyreferred to herein as “vectors”) by a variety of procedures. In general,the DNA sequence is inserted into an appropriate restrictionendonuclease site(s) by standard procedures. Such procedures and relatedsub-cloning procedures are deemed to be within the scope of knowledge ofthose skilled in the art.

Recombinant filamentous fungi comprising the coding sequence for adesired cellulase may be produced by introducing a heterologous nucleicacid construct comprising the desired cellulase coding sequence into thecells of a selected strain of the filamentous fungi.

Once the desired form of a desired cellulase nucleic acid sequence isobtained, it may be modified in a variety of ways. Where the sequenceinvolves non-coding flanking regions, the flanking regions may besubjected to resection, mutagenesis, etc. Thus, transitions,transversions, deletions, and insertions may be performed on thenaturally occurring sequence.

A selected desired cellulase coding sequence may be inserted into asuitable vector according to well-known recombinant techniques and usedto transform filamentous fungi capable of cellulase expression. Due tothe inherent degeneracy of the genetic code, other nucleic acidsequences which encode substantially the same or a functionallyequivalent amino acid sequence may be used to clone and express adesired cellulase. Therefore it is appreciated that such substitutionsin the coding region fall within the sequence variants covered by thepresent invention.

The present invention also includes recombinant nucleic acid constructscomprising one or more of the desired cellulase-encoding nucleic acidsequences as described above. The constructs comprise a vector, such asa plasmid or viral vector, into which a sequence of the invention hasbeen inserted, in a forward or reverse orientation.

Heterologous nucleic acid constructs may include the coding sequence fora desired cellulase: (I) in isolation; (ii) in combination withadditional coding sequences; such as fusion protein or signal peptidecoding sequences, where the desired cellulase coding sequence is thedominant coding sequence; (iii) in combination with non-codingsequences, such as introns and control elements, such as promoter andterminator elements or 5 and/or 3′ untranslated regions, effective forexpression of the coding sequence in a suitable host; and/or (iv) in avector or host environment in which the desired cellulase codingsequence is a heterologous gene.

In one aspect of the present invention, a heterologous nucleic acidconstruct is employed to transfer a desired cellulase-encoding nucleicacid sequence into a cell in vitro, with established filamentous fungaland yeast lines preferred. For long-term, production of a desiredcellulase, stable expression is preferred. It follows that any methodeffective to generate stable transformants may be used in practicing theinvention.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as promoter and termination sequences. The vectormay comprise regulatory sequences, including, for example, non-codingsequences, such as introns and control elements, i.e., promoter andterminator elements or 5′ and/or 3′ untranslated regions, effective forexpression of the coding sequence in host cells (and/or in a vector orhost cell environment in which a modified soluble protein antigen codingsequence is not normally expressed), operably linked to the codingsequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, many of which are commercially availableand/or are described in Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and induciblepromoters, examples of which include a CMV promoter, an SV40 earlypromoter, an RSV promoter, an EF-1α promoter, a promoter containing thetet responsive element (TRE) in the tet-on or tet-off system asdescribed (ClonTech and BASF), the beta actin promoter and themetallothionine promoter that can upregulated by addition of certainmetal salts. A promoter sequence is a DNA sequence which is recognizedby the particular filamentous fungus for expression purposes. It isoperably linked to DNA sequence encoding a variant CBH1 polypeptide.Such linkage comprises positioning of the promoter with respect to theinitiation codon of the DNA sequence encoding the variant CBH1polypeptide in the disclosed expression vectors. The promoter sequencecontains transcription and translation control sequence which mediatethe expression of the variant CBH1 polypeptide. Examples include thepromoters from the Aspergillus niger, A. awamori or A. oryzaeglucoamylase, alpha-amylase, or alpha-glucosidase encoding genes; the A.nidulans gpdA or trpC Genes; the Neurospora crassa cbh1 or trp1 genes;the A. niger or Rhizomucor miehei aspartic proteinase encoding genes;the H. jecorina cbh1, cbh2, egl1, egl2, or other cellulase encodinggenes.

The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.Typical selectable marker genes include argB from A. nidulans or H.jecorina, amdS from A. nidulans, pyr4 from Neurospora crassa or H.jecorina, pyrG from Aspergillus niger or A. nidulans. Additionalexemplary selectable markers include, but are not limited to trpc, trp1,oliC31, niaD or leu2, which are included in heterologous nucleic acidconstructs used to transform a mutant strain such as trp-, pyr-, leu-and the like.

Such selectable markers confer to transformants the ability to utilize ametabolite that is usually not metabolized by the filamentous fungi. Forexample, the amdS gene from H. jecorina which encodes the enzymeacetamidase that allows transformant cells to grow on acetamide as anitrogen source. The selectable marker (e.g. pyrG) may restore theability of an auxotrophic mutant strain to grow on a selective minimalmedium or the selectable marker (e.g. olic31) may confer totransformants the ability to grow in the presence of an inhibitory drugor antibiotic.

The selectable marker coding sequence is cloned into any suitableplasmid using methods generally employed in the art. Exemplary plasmidsinclude pUC18, pBR322, pRAX and pUC100. The pRAX plasmid contains AMA1sequences from A. nidulans, which make it possible to replicate in A.niger.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook et al., 1989; Freshney, 1987; Ausubel, et al., 1993; andColigan et al., 1991. All patents, patent applications, articles andpublications mentioned herein, are hereby expressly incorporated hereinby reference.

B. Host Cells and Culture Conditions for CBH1 Production

(i) Filamentous Fungi

Thus, the present invention provides filamentous fungi comprising cellswhich have been modified, selected and cultured in a manner effective toresult in desired cellulase production or expression relative to thecorresponding non-transformed parental fungi.

Examples of species of parental filamentous fungi that may be treatedand/or modified for desired cellulase expression include, but are notlimited to Trichoderma, Penicillium sp., Humicola sp., includingHumicola insolens; Aspergillus sp., including Aspergillus niger,Chrysosporium sp., Fusarium sp., Hypocrea sp., and Emericella sp.

Cells expressing a desired cellulase are cultured under conditionstypically employed to culture the parental fungal line. Generally, cellsare cultured in a standard medium containing physiological salts andnutrients, such as described in Pourquie, J. et al., Biochemistry andGenetics of Cellulose Degradation, eds. Aubert, J. P. et al., AcademicPress, pp. 71-86, 1988 and Ilmen, M. et al., Appl. Environ. Microbiol.63:1298-1306, 1997. Culture conditions are also standard, e.g., culturesare incubated at 28° C. in shaker cultures or fermenters until desiredlevels of desired cellulase expression are achieved.

Preferred culture conditions for a given filamentous fungus may be foundin the scientific literature and/or from the source of the fungi such asthe American Type Culture Collection (ATCC). After fungal growth hasbeen established, the cells are exposed to conditions effective to causeor permit the expression of a desired cellulase.

In cases where a desired cellulase coding sequence is under the controlof an inducible promoter, the inducing agent, e.g., a sugar, metal saltor antibiotics, is added to the medium at a concentration effective toinduce desired cellulase expression.

In one embodiment, the strain comprises Aspergillus niger, which is auseful strain for obtaining overexpressed protein. For example A. nigervar awamori dgr246 is known to secrete elevated amounts of secretedcellulases (Goedegebuur et al, Curr. Genet (2002) 41: 89-98). Otherstrains of Aspergillus niger var awamori such as GCDAP3, GCDAP4 andGAP3-4 are known Ward et al (Ward, M, Wilson, L. J. and Kodama, K. H.,1993, Appl. Microbiol. Biotechnol. 39:738-743).

In another embodiment, the strain comprises Trichoderma reesei, which isa useful strain for obtaining overexpressed protein. For example,RL-P37, described by Sheir-Neiss, et al., Appl. Microbiol. Biotechnol.20:46-53 (1984) is known to secrete elevated amounts of cellulaseenzymes. Functional equivalents of RL-P37 include Trichoderma reeseistrain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). Itis contemplated that these strains would also be useful inoverexpressing variant CBH1.

Where it is desired to obtain the desired cellulase in the absence ofpotentially detrimental native cellulolytic activity, it is useful toobtain a host cell strain which has had one or more cellulase genesdeleted prior to introduction of a DNA construct or plasmid containingthe DNA fragment encoding the desired cellulase. Such strains may beprepared by the method disclosed in U.S. Pat. No. 5,246,853 and WO92/06209, which disclosures are hereby incorporated by reference. Byexpressing a desired cellulase in a host microorganism that is missingone or more cellulase genes, the identification and subsequentpurification procedures are simplified.

Gene deletion may be accomplished by inserting a form of the desiredgene to be deleted or disrupted into a plasmid by methods known in theart. The deletion plasmid is then cut at an appropriate restrictionenzyme site(s), internal to the desired gene coding region, and the genecoding sequence or part thereof replaced with a selectable marker.Flanking DNA sequences from the locus of the gene to be deleted ordisrupted, preferably between about 0.5 to 2.0 kb, remain on either sideof the selectable marker gene. An appropriate deletion plasmid willgenerally have unique restriction enzyme sites present therein to enablethe fragment containing the deleted gene, including flanking DNAsequences, and the selectable marker gene to be removed as a singlelinear piece.

A selectable marker must be chosen so as to enable detection of thetransformed microorganism. Any selectable marker gene that is expressedin the selected microorganism will be suitable. For example, withAspergillus sp., the selectable marker is chosen so that the presence ofthe selectable marker in the transformants will not significantly affectthe properties thereof. Such a selectable marker may be a gene thatencodes an assayable product. For example, a functional copy of aAspergillus sp. gene may be used which if lacking in the host strainresults in the host strain displaying an auxotrophic phenotype.

In a preferred embodiment, a pyrG-derivative strain of Aspergillus sp.is transformed with a functional pyrG gene, which thus provides aselectable marker for transformation. A pyrG-derivative strain may beobtained by selection of Aspergillus sp. strains that are resistant tofluoroorotic acid (FOA). The pyrG gene encodesorotidine-5′-monophosphate decarboxylase, an enzyme required for thebiosynthesis of uridine. Strains with an intact pyrG gene grow in amedium lacking uridine but are sensitive to fluoroorotic acid. It ispossible to select pyrG-derivative strains that lack a functionalorotidine monophosphate decarboxylase enzyme and require uridine forgrowth by selecting for FOA resistance. Using the FOA selectiontechnique it is also possible to obtain uridine-requiring strains whichlack a functional orotate pyrophosphoribosyl transferase. It is possibleto transform these cells with a functional copy of the gene encodingthis enzyme (Berges & Barreau, Curr. Genet. 19:359-365 (1991), and vanHartingsveldt et al., (1987) Development of a homologous transformationsystem for Aspergillus niger based on the pyrG gene. Mol. Gen. Genet.206:71-75). Selection of derivative strains is easily performed usingthe FOA resistance technique referred to above, and thus, the pyrG geneis preferably employed as a selectable marker.

To transform pyrG⁻ Aspergillus sp. so as to be lacking in the ability toexpress one or more cellulase genes, a single DNA fragment comprising adisrupted or deleted cellulase gene is then isolated from the deletionplasmid and used to transform an appropriate pyr⁻ Aspergillus host.Transformants are then identified and selected based on their ability toexpress the pyrG gene product and thus compliment the uridine auxotrophyof the host strain. Southern blot analysis is then carried out on theresultant transformants to identify and confirm a double crossoverintegration event that replaces part or all of the coding region of thegenomic copy of the gene to be deleted with the pyr4 selectable markers.

Although the specific plasmid vectors described above relate topreparation of pyr⁻ transformants, the present invention is not limitedto these vectors. Various genes can be deleted and replaced in theAspergillus sp. strain using the above techniques. In addition, anyavailable selectable markers can be used, as discussed above. In fact,any Aspergillus sp. gene that has been cloned, and thus identified, canbe deleted from the genome using the above-described strategy.

As stated above, the host strains used are derivatives of Aspergillussp. that lack or have a nonfunctional gene or genes corresponding to theselectable marker chosen. For example, if the selectable marker of pyrGis chosen, then a specific pyrG⁻ derivative strain is used as arecipient in the transformation procedure. Similarly, selectable markerscomprising Aspergillus sp. genes equivalent to the Aspergillus nidulansgenes amdS, argB, trpC, niaD may be used. The corresponding recipientstrain must therefore be a derivative strain such as argB⁻, trpC⁻,niaD⁻, respectively.

DNA encoding the desired cellulase is then prepared for insertion intoan appropriate microorganism. According to the present invention, DNAencoding a desired cellulase comprises the DNA necessary to encode for aprotein that has functional cellulolytic activity. The DNA fragmentencoding the desired cellulase may be functionally attached to a fungalpromoter sequence, for example, the promoter of the glaA gene.

It is also contemplated that more than one copy of DNA encoding adesired cellulase may be recombined into the strain to facilitateoverexpression. The DNA encoding the desired cellulase may be preparedby the construction of an expression vector carrying the DNA encodingthe cellulase. The expression vector carrying the inserted DNA fragmentencoding the desired cellulase may be any vector which is capable ofreplicating autonomously in a given host organism or of integrating intothe DNA of the host, typically a plasmid. In preferred embodiments twotypes of expression vectors for obtaining expression of genes arecontemplated. The first contains DNA sequences in which the promoter,gene-coding region, and terminator sequence all originate from the geneto be expressed. Gene truncation may be obtained where desired bydeleting undesired DNA sequences (e.g., coding for unwanted domains) toleave the domain to be expressed under control of its owntranscriptional and translational regulatory sequences. A selectablemarker is also contained on the vector allowing the selection forintegration into the host of multiple copies of the novel genesequences.

The second type of expression vector is preassembled and containssequences required for high-level transcription and a selectable marker.It is contemplated that the coding region for a gene or part thereof canbe inserted into this general-purpose expression vector such that it isunder the transcriptional control of the expression cassettes promoterand terminator sequences. For example, pRAX is such a general-purposeexpression vector. Genes or part thereof can be inserted downstream ofthe strong gleA promoter.

In the vector, the DNA sequence encoding the desired cellulase of thepresent invention should be operably linked to transcriptional andtranslational sequences, i.e., a suitable promoter sequence and signalsequence in reading frame to the structural gene. The promoter may beany DNA sequence that shows transcriptional activity in the host celland may be derived from genes encoding proteins either homologous orheterologous to the host cell. An optional signal peptide provides forextracellular production of the desired cellulase. The DNA encoding thesignal sequence is preferably that which is naturally associated withthe gene to be expressed, however the signal sequence from any suitablesource is contemplated in the present invention.

The procedures used to fuse the DNA sequences coding for the desiredcellulase of the present invention with the promoter into suitablevectors are well known in the art.

The DNA vector or construct described above may be introduced in thehost cell in accordance with known techniques such as transformation,transfection, microinjection, microporation, biolistic bombardment andthe like.

The preferred method in the present invention to prepare Aspergillus sp.for transformation involves the preparation of protoplasts from fungalmycelium. See Campbell et al., Improved transformation efficiency of A.niger using homologous niaD gene for nitrate reductase. Curr. Genet.16:53-56; 1989. The mycelium can be obtained from germinated vegetativespores. The mycelium is treated with an enzyme that digests the cellwall resulting in protoplasts. The protoplasts are then protected by thepresence of an osmotic stabilizer in the suspending medium. Thesestabilizers include sorbitol, mannitol, potassium chloride, magnesiumsulfate and the like. Usually the concentration of these stabilizersvaries between 0.8 M and 1.2 M. It is preferable to use about a 1.2 Msolution of sorbitol in the suspension medium.

Uptake of the DNA into the host Aspergilius sp. strain is dependent uponthe calcium ion concentration. Generally between about 10 mM CaCl₂ and50 mM CaCl₂ is used in an uptake solution. Besides the need for thecalcium ion in the uptake solution, other items generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesuionic acid) and polyethyleneglycol (PEG). It is believed that the polyethylene glycol acts to fusethe cell membranes thus permitting the contents of the medium to bedelivered into the cytoplasm of the Aspergillus sp. strain and theplasmid DNA is transferred to the nucleus. This fusion frequently leavesmultiple copies of the plasmid DNA tenderly integrated into the hostchromosome.

Usually a suspension containing the Aspergillus sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁶/mL, preferably 2×10⁵/mL are used in transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol; 50 mM CaCl₂) are mixed with the desired DNA. Generally ahigh concentration of PEG is added to the uptake solution. From 0.1 to 1volume of 25% PEG 4000 can be added to the protoplast suspension.However, it is preferable to add about 0.25 volumes to the protoplastsuspension. Additives such as dimethyl sulfoxide, heparin, spermidine,potassium chloride and the like may also be added to the uptake solutionand aid in transformation.

Generally, the mixture is then incubated at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired gene or DNAsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is preferably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then incubated either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. This growth medium permits the growth of transformantsonly. Any growth medium can be used in the present invention that issuitable to grow the desired transformants. However, if Pyr⁺transformants are being selected it is preferable to use a growth mediumthat contains no uridine. The subsequent colonies are transferred andpurified on a growth medium depleted of uridine.

At this stage, stable transformants may be distinguished from unstabletransformants by their faster growth rate and the formation of circularcolonies with a smooth, rather than ragged outline on solid culturemedium lacking uridine. Additionally, in some cases a further test ofstability may made by growing the transformants on solid non-selectivemedium (i.e. containing uridine), harvesting spores from this culturemedium and determining the percentage of these spores which willsubsequently germinate and grow on selective medium lacking uridine.

In a particular embodiment of the above method, the desired cellulase(s)are recovered in active form from the host cell after growth in liquidmedia either as a result of the appropriate post translationalprocessing of the desired cellulase.

(ii) Yeast

The present invention also contemplates the use of yeast as a host cellfor desired cellulase production. Several other genes encodinghydrolytic enzymes have been expressed in various strains of the yeastS. cerevisiae. These include sequences encoding for two endoglucanases(Penttila et al., 1987), two cellobiohydrolases (Penttila et al., 1988)and one beta-glucosidase from Trichoderma reesei (Cummings and Fowler,1996), a xylanase from Aureobasidlium pullulans (Li and Ljungdahl,1996), an alpha-amylase from wheat (Rothstein et al., 1987), etc. Inaddition, a cellulase gene cassette encoding the Butyrivibriofibrisolvens endo-[beta]-1,4-glucanase (END1), Phanerochaetechrysospodium cellobiohydrolase (CBH1), the Ruminococcus flavefacienscellodextrinase (CEL1) and the Endomyces fibrilizer cellobiase (Bgl1)was successfully expressed in a laboratory strain of S. cerevisiae (VanRensburg et al., 1998).

C. Introduction of a Desired Cellulase-Encoding Nucleic Acid Sequenceinto Host Cells.

The invention further provides cells and cell compositions which havebeen genetically modified to comprise an exogenously provided desiredcellulase-encoding nucleic acid sequence. A parental cell or cell linemay be genetically modified (i.e., transduced, transformed ortransfected) with a cloning vector or an expression vector. The vectormay be, for example, in the form of a plasmid, a viral particle, aphage, etc, as further described above.

The methods of transformation of the present invention may result in thestable integration of all or part of the transformation vector into thegenome of the filamentous fungus. However, transformation resulting inthe maintenance of a self-replicating extra-chromosomal transformationvector is also contemplated.

Many standard transfection methods can be used to produce Trichodermareesei cell lines that express large quantities of the heterologusprotein. Some of the published methods for the introduction of DNAconstructs into cellulase-producing strains of Trichoderma includeLorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356;Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174;Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 61:155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl.Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila andKolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, forStreptomyces Hopwood et al., 1986, The John Innes Foundation, Norwich,UK and for Bacillus, Brigidi, DeRossi, Bertarini, Riccardi andMatteuzzi, 1990, FEMS Microbiol. Lett. 67: 135-138, 1990.

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra). Also of use is theAgrobacterium-mediated transfection method described in U.S. Pat. No.6,255,115. It is only necessary that the particular genetic engineeringprocedure used be capable of successfully introducing at least one geneinto the host cell capable of expressing the heterologous gene.

In addition, heterologous nucleic acid constructs comprising a desiredcellulase-encoding nucleic acid sequence can be transcribed in vitro,and the resulting RNA introduced into the host cell by well-knownmethods, e.g., by injection.

The invention further includes novel and useful transformants offilamentous fungi such as H. jecorina and A. niger for use in producingfungal cellulase compositions. The invention includes transformants offilamentous fungi especially fungi comprising the desired cellulasecoding sequence, or deletion of the endogenous cbh coding sequence.

VII. ANALYSIS FOR CBH1 NUCLEIC ACID CODING SEQUENCES AND/OR PROTEINEXPRESSION

In order to evaluate the expression of a desired cellulase by a cellline that has been transformed with a desired cellulase-encoding nucleicacid construct, assays can be carried out at the protein level, the RNAlevel or by use of functional bioassays particular to cellobiohydrolaseactivity and/or production.

In one exemplary application of the desired cellulase nucleic acid andprotein sequences described herein, a genetically modified strain offilamentous fungi, e.g., Trichoderma reesei, is engineered to produce anincreased amount of a desired cellulase. Such genetically modifiedfilamentous fungi would be useful to produce a cellulase product withgreater increased cellulolytic capacity. In one approach, this isaccomplished by introducing the coding sequence for a desired cellulaseinto a suitable host, e.g., a filamentous fungi such as Aspergillusniger.

Accordingly, the invention includes methods for expressing a desiredcellulase in a filamentous fungus or other suitable host by introducingan expression vector containing the DNA sequence encoding a desiredcellulase into cells of the filamentous fungus or other suitable host.

In another aspect, the invention includes methods for modifying theexpression of a desired cellulase in a filamentous fungus or othersuitable host. Such modification includes a decrease or elimination inexpression of the endogenous CBH.

In general, assays employed to analyze the expression of a desiredcellulase include, Northern blotting, dot blotting (DNA or RNAanalysis), RT-PCR (reverse transcriptase polymerase chain reaction), orin situ hybridization, using an appropriately labeled probe (based onthe nucleic acid coding sequence) and conventional Southern blotting andautoradiography.

In addition, the production and/or expression of a desired cellulase maybe measured in a sample directly, for example, by assays forcellobiohydrolase activity, expression and/or production. Such assaysare described, for example, in Becker et al., Biochem J. (2001)356:19-30 and Mitsuishi et al., FEBS (1990) 275:135-138, each of whichis expressly incorporated by reference herein. The ability of CBH1 tohydrolyze isolated soluble and insoluble substrates can be measuredusing assays described in Srisodsuk et al., J. Biotech. (1997) 57:4957and Nidetzky and Claeyssens Biotech. Bioeng. (1994) 44:961-966.Substrates useful for assaying cellobiohydrolase, endoglucanase orβ-glucosidase activities include crystalline cellulose, filter paper,phosphoric acid swollen cellulose, cellooligosaccharides,methylumbelliferyl lactoside, methylumbelliferyl cellobioside,orthonitrophenyl lactoside, paranitrophenyl lactoside, orthonitrophenylcellobioside, paranitrophenyl cellobioside.

In addition, protein expression, may be evaluated by immunologicalmethods, such as immunohistochemical staining of cells, tissue sectionsor immunoassay of tissue culture medium, e.g., by Western blot or ELISA.Such immunoassays can be used to qualitatively and quantitativelyevaluate expression of a desired cellulase. The details of such methodsare known to those of skill in the art and many reagents for practicingsuch methods are commercially available.

A purified form of a desired cellulase may be used to produce eithermonoclonal or polyclonal antibodies specific to the expressed proteinfor use in various immunoassays. (See, e.g., Hu et al., 1991). Exemplaryassays include ELISA, competitive immunoassays, radioimmunoassays,Western blot, indirect immunofluorescent assays and the like. Ingeneral, commercially available antibodies and/or kits may be used forthe quantitative immunoassay of the expression level ofcellobiohydrolase proteins.

VIII. ISOLATION AND PURIFICATION OF RECOMBINANT CBH1 PROTEIN

In general, a desired cellulase protein produced in cell culture issecreted into the medium and may be purified or isolated, e.g., byremoving unwanted components from the cell culture medium. However, insome cases, a desired cellulase protein may be produced in a cellularform necessitating recovery from a cell lysate. In such cases thedesired cellulase protein is purified from the cells in which it wasproduced using techniques routinely employed by those of skill in theart. Examples include, but are not limited to, affinity chromatography(Van Tilbeurgh et al., 1984), ion-exchange chromatographic methods(Goyal et al., 1991; Fliess et al., 1983; Bhikhabhai et al., 1984;Ellouz et al., 1987), including ion-exchange using materials with highresolution power (Medve et al., 1998), hydrophobic interactionchromatography (Tomaz and Queiroz, 1999), and two-phase partitioning(Brumbauer, et al., 1999).

Typically, the desired cellulase protein is fractionated to segregateproteins having selected properties, such as binding affinity toparticular binding agents, e.g., antibodies or receptors; or which havea selected molecular weight range, or range of isoelectic points.

Once expression of a given desired cellulase protein is achieved, thedesired cellulase protein thereby produced is purified from the cells orcell culture. Exemplary procedures suitable for such purificationinclude the following: antibody-affinity column chromatography, ionexchange chromatography; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and gelfiltration using, e.g., Sephadex G-75. Various methods of proteinpurification may be employed and such methods are known in the art anddescribed e.g. in Deutscher, 1990; Scopes, 1982. The purificationstep(s) selected will depend, e.g., on the nature of the productionprocess used and the particular protein produced.

IX. UTILITY OF cbh1 And CBH1

It can be appreciated that the desired cellulase nucleic acids, thedesired cellulase protein and compositions comprising desired cellulaseprotein activity find utility in a wide variety applications, some ofwhich are described below.

New and improved cellulase compositions that comprise varying amounts ofa desired cellulase find utility in detergent compositions that exhibitenhanced cleaning ability, function as a softening agent and/or improvethe feel of cotton fabrics (e.g., “stone washing” or “biopolishing”), incompositions for degrading wood pulp into sugars (e.g., for bio-ethanolproduction), and/or in feed compositions. The isolation andcharacterization of cellulase of each type provides the ability tocontrol the aspects of such compositions.

Desired cellulases with decreased thermostability find uses, forexample, in areas where the enzyme activity is required to beneutralized at lower temperatures so that other enzymes that may bepresent are left unaffected. In addition, the enzymes may find utilityin the limited conversion of cellulosics, for example, in controllingthe degree of crystallinity or of cellulosic chain-length. Afterreaching the desired extent of conversion the saccharifying temperaturecan be raised above the survival temperature of the de-stabilized CBH1.As the CBH1 activity is essential for hydrolysis of crystallinecellulose, conversion of crystalline cellulose will cease at theelevated temperature.

In one approach, the cellulase of the invention finds utility indetergent compositions or in the treatment of fabrics to improve thefeel and appearance.

Since the rate of hydrolysis of cellulosic products may be increased byusing a transformant having at least one additional copy of the desiredcellulase gene, either as a replicative plasmid or inserted into thegenome, products that contain cellulose or heteroglycans can be degradedat a faster rate and to a greater extent. Products made from cellulosesuch as paper, cotton, cellulosic diapers and the like can be degradedmore efficiently in a landfill. Thus, the fermentation productobtainable from the transformants or the transformants alone may be usedin compositions to help degrade by liquefaction a variety of celluloseproducts that add to the overcrowded landfills.

Separate saccharification and fermentation is a process wherebycellulose present in biomass, e.g., corn stover, is converted to glucoseand subsequently yeast strains convert glucose into ethanol.Simultaneous saccharification and fermentation is a process wherebycellulose present in biomass, e.g., corn stover, is converted to glucoseand, at the same time and in the same reactor, yeast strains convertglucose into ethanol. Thus, in another approach, the desired cellulaseof the invention finds utility in the degradation of biomass to ethanol.Ethanol production from readily available sources of cellulose providesa stable, renewable fuel source.

Cellulose-based feedstocks are comprised of agricultural wastes, grassesand woods and other low-value biomass such as municipal waste (e.g.,recycled paper, yard clippings, etc.). Ethanol may be produced from thefermentation of any of these cellulosic feedstocks. However, thecellulose must first be converted to sugars before there can beconversion to ethanol.

A large variety of feedstocks may be used with the inventive desiredcellulase(s) and the one selected for use may depend on the region wherethe conversion is being done. For example, in the Midwestern UnitedStates agricultural wastes such as wheat straw, corn stover and bagassemay predominate while in California rice straw may predominate. However,it should be understood that any available cellulosic biomass may beused in any region.

A cellulase composition containing an enhanced amount ofcellobiohydrolase finds utility in ethanol production. Ethanol from thisprocess can be further used as an octane enhancer or directly as a fuelin lieu of gasoline which is advantageous because ethanol as a fuelsource is more environmentally friendly than petroleum derived products.It is known that the use of ethanol will improve air quality andpossibly reduce local ozone levels and smog. Moreover, utilization ofethanol in lieu of gasoline can be of strategic importance in bufferingthe impact of sudden shifts in non-renewable energy and petro-chemicalsupplies.

Ethanol can be produced via saccharification and fermentation processesfrom cellulosic biomass such as trees, herbaceous plants, municipalsolid waste and agricultural and forestry residues. However, the ratioof individual cellulase enzymes within a naturally occurring cellulasemixture produced by a microbe may not be the most efficient for rapidconversion of cellulose in biomass to glucose. It is known thatendoglucanases act to produce new cellulose chain ends which themselvesare substrates for the action of cellobiohydrolases and thereby improvethe efficiency of hydrolysis of the entire cellulase system. Therefore,the use of increased or optimized cellobiohydrolase activity may greatlyenhance the production of ethanol.

Thus, the inventive cellobiohydrolase(s) finds use in the hydrolysis ofcellulose to its sugar components. In one embodiment, a desiredcellulase is added to the biomass prior to the addition of afermentative organism. In a second embodiment, a desired cellulase isadded to the biomass at the same time as a fermentative organism.Optionally, there may be other cellulase components present in eitherembodiment.

In another embodiment the cellulosic feedstock may be pretreated.Pretreatment may be by elevated temperature and the addition of eitherof dilute acid, concentrated acid or dilute alkali solution. Thepretreatment solution is added for a time sufficient to at leastpartially hydrolyze the hemicellulose components and then neutralized.

The detergent compositions of this invention may employ besides thecellulase composition (irrespective of the cellobiohydrolase content,i.e., cellobiohydrolase-free, substantially cellobiohydrolase-free, orcellobiohydrolase enhanced), a surfactant, including anionic, non-ionicand ampholytic surfactants, a hydrolase, building agents, bleachingagents, bluing agents and fluorescent dyes, caking inhibitors,solubilizers, cationic surfactants and the like. All of these componentsare known in the detergent art. The cellulase composition as describedabove can be added to the detergent composition either in a liquiddiluent, in granules, in emulsions, in gels, in pastes, and the like.Such forms are well known to the skilled artisan. When a solid detergentcomposition is employed, the cellulase composition is preferablyformulated as granules. Preferably, the granules can be formulated so asto contain a cellulase protecting agent. For a more thorough discussion,see U.S. Pat. No. 6,162,782 entitled “Detergent compositions containingcellulase compositions deficient in CBH1 type components,” which isincorporated herein by reference.

Preferably the cellulase compositions are employed from about 0.00005weight percent to about 5 weight percent relative to the total detergentcomposition. More preferably, the cellulase compositions are employedfrom about 0.0002 weight percent to about 2 weight percent relative tothe total detergent composition.

In addition the desired cellulase nucleic acid sequence finds utility inthe identification and characterization of related nucleic acidsequences. A number of techniques useful for determining (predicting orconfirming) the function of related genes or gene products include, butare not limited to, (A) DNA/RNA analysis, such as (1) overexpression,ectopic expression, and expression in other species; (2) gene knock-out(reverse genetics, targeted knock-out, viral induced gene silencing(VIGS, see Baulcombe, 1999); (3) analysis of the methylation status ofthe gene, especially flanking regulatory regions; and (4) in situhybridization; (B) gene product analysis such as (1) recombinant proteinexpression; (2) antisera production, (3) immunolocalization; (4)biochemical assays for catalytic or other activity; (5) phosphorylationstatus; and (6) interaction with other proteins via yeast two-hybridanalysis; (C) pathway analysis, such as placing a gene or gene productwithin a particular biochemical or signaling pathway based on itsoverexpression phenotype or by sequence homology with related genes; and(D) other analyses which may also be performed to determine or confirmthe participation of the isolated gene and its product in a particularmetabolic or signaling pathway, and help determine gene function.

EXAMPLES

The present invention is described in further detain in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein.

Example 1 Identification of CBH1 Homologs

This example illustrates the novel CBH1 homologs found in a variety offungi. Genomic DNA was prepared for several different microorganisms forthe purpose of undertaking a PCR reaction to determine whetherhomologous CBH1 cellulases are encoded by the DNA of a particularorganism.

Isolation of Genomic DNA

Genomic DNA may be isolated using any method known in the art. In thisset of experiments we received 48 genomic DNA solutions from diverseHypocrea and Trichoderma species from collaboration with the TechnicalUniversity of Vienna (TUV), Hypocrea schweinitzii (CBS 243.63), Hypocreaorientalis (PPR13894), Trichoderma pseudokoningii (CBS 408.91) andTrichoderma konilangbra (isolate 1). However, the following protocol maybe used:

Cells are grown at 30° C. in 20 ml Potato Dextrose Broth (PDB) for 24hours. The cells are diluted 1:20 in fresh PDB medium and grownovernight. Two milliliters of cells are centrifuged and the pelletwashed in 1 ml KC (60 g KCl, 2 g citric acid per liter, pH adjusted to6.2 with 1 M KOH). The cell pellet is resuspended in 900 μl KC. 100 μl(20 mg/ml) Novozyme® is added, mixed gently and the protoplastationfollowed microscopically at 37° C. until greater than 95% protoplastsare formed for a maximum of 2 hours. The cells are centrifuged at 1500rpm (460 g) for 10 minutes. 200 μl TES/SDS (10 mM Tris, 50 mM EDTA, 150mM NaCl, 1% SDS) is added, mixed and incubated at room temperature for 5minutes. DNA is isolated using a Qiagen mini-prep isolation kit(Qiagen). The column is eluted with 100 μl milli-Q water and the DNAcollected.

An alternative method using the FastPrep® method may be desirable. Thesystem consists of the FastPrep® Instrument as well as FastPrep® kitsfor nucleic acid isolation. FastPrep® is available from Obiogene.

Construction of Primers

PCR was performed on a standard PCR machine such as the PCT-200 PeltierThermal Cycler from MJ Research Inc. under the following conditions:

-   -   1) 1 minute at 96° C. for 1 cycle    -   2) 30 seconds at 94° C.        -   90 seconds at 45° C. (+1° C. per cycle)        -   2 minutes at 72° C.    -   3) Repeat step 2 for 10 cycles    -   4) 30 seconds at 94° C.        -   90 seconds at 55° C.        -   2 minutes at 72° C.    -   5) Repeat step 4 for 20 cycles    -   6) 7 minutes at 72° C. for 1 cycle, and    -   7) lower temperature to 15° C. for storage and further analysis.

The following DNA primers were constructed for use in amplification ofhomologous CBH1 genes from genomic DNA's isolated from variousmicroorganisms. All symbols used herein for protein and DNA sequencescorrespond to IUPAC IUB Biochemical Nomenclature Commission codes.

Homologous 5′ (FRG192) and 3′ (FRG193) primers were developed based onthe sequence of CBH1 from Trichoderma reesei. Both primers containedGateway cloning sequences from Invitrogen® at the 5′ of the primer.Primer FRG192 contained attB1 sequence and primer FRG193 contained attB2sequence.

Sequence of FRG192 without the attB1:

ATGTATCGGAAGTTGGCCG (signal sequence of CBH1 H. jecorina)

Sequence of FRG193 without the attB2:

TTACAGGCACTGAGAGTAG (cellulose binding module of CBH1 H. jecorina)

PCR conditions were as follows: 10 μL of 10× reaction buffer (10×reaction buffer comprising 100 mM Tris HCl, pH 8-8.5; 250 mM KCl; 50 mM(NH₄)SO₄; 20 mM MgSO₄); 0.2 mM each of dATP, dTTP, dGTP, dCTP (finalconcentration), 1 μL of 100 ng/μL genomic DNA, 0.5 μL of PWO polymerase(Boehringer Mannheim, Cat # 1644-947) at 1 unit per μL, 0.2 μM of eachprimer, FRG192 and FRG193, (final concentration), 4 μl DMSO and water to100 μL.

These conditions finally resulted in 4 genes from different species:

1. Hypocrea schweinitzii (CBS 243.63)

2. Hypocrea orientalis (PPR13894)

3. Trichoderma pseudokoningii (CBS 408.91)

4. Trichoderma konilangbra

Isolation of Cel7A Gene Sequences

The full length sequences were obtained directly by using the N terminal(FRG192) and C terminal (FRG193) primers. The full length DNA sequenceswere translated into three open reading frames using Vector NTIsoftware. Comparison of DNA and protein sequences to H. jecorina Cel7Awere performed to identify the putative intron sequences. Translation ofthe genomic DNA sequence without the intron sequences revealed theprotein sequence of homologous CBH1's. Full length genes have beenobtained and are provided in FIGS. 3, 5, 7 and 9.

Example 2 Expression and Thermostability of CBH1 Homologs

The full-length genes from Example 1 were transferred to the A. nigerGateway compatible destination vector, which was developed by Genencor.This vector was built by using the pRAX1 as a backbone, shown in FIG.11, according to the manual given in Gateway™ Cloning Technology:version 1 page 34-38.

The newly developed expression vector is shown in FIG. 12; this is aproduct of transferring the new genes into the destination vectorpRAXdes2. This resulted in the final expression vectors calledpRAXdesCBH1(specified with the species name)

The constructs has been transformed into A. niger var. awamori accordingto the method described by Cao et al (Cao Q-N, Stubbs M, Ngo K Q P, WardM, Cunningham A, Pai E F, Tu G-C and Hofmann T (2000)Penicillopepsin-JT2 a recombinant enzyme from Penicillium janthinellumand contribution of a hydrogen bond in subsite S3 to kcat ProteinScience 9:991-1001).

Transformants were streaked on minimal medium plates (Ballance D J,Buxton F P, and Turner G (1983) Transformation of Aspergillus nidulansby the orotidine-5′-phosphate decarboxylase gene of Neurospora crassaBiochem Biophys Res Commun 112:284-289) and grown for 4 days at 30° C.Spores were collected using methods well known in the art. A. nidulansconidia are harvested in water (by rubbing the surface of a conidiatingculture with a sterile bent glass rod to dislodge the spores) and can bestored for weeks to months at 4° C. without a serious loss of viability.However, freshly harvested spores germinate more reproducibly. Forlong-term storage, spores can be stored in 50% glycerol at −20° C., orin 15-20% glycerol at −80° C. Glycerol is more easily pipetted as an 80%solution in water. 800 μl of aqueous conidial suspension (as made for 4°C. storage) added to 200 μl 80% glycerol is used for a −80° C. stock;400 μl suspension added to 600 μl 80% glycerol is used for a −20° C.stock. Vortex before freezing. For mutant collections, small pieces ofconidiating cultures can be excised and placed in 20% glycerol,vortexed, and frozen as −80° C. stocks. In our case we store them in 50%glycerol at −80° C.

A. niger var awamori transformants were grown on minimal medium lackinguridine (Ballance et al. 1983). Transformants were screened forcellulase activity by inoculating 1 cm² of spore suspension from thesporulated grown agar plate into 100 ml shake flasks for 3 days at 37°C. as described by Cao et al. (2000).

The CBH1 activity assay is based on the hydrolysis of the nonfluorescent4-methylumbelliferyl-β-lactoside to the products lactose and7-hydroxy-4-methylcoumarin, the latter product is responsible for thefluorescent signal. Pipette 170 μl 50 mM NaAc buffer pH 4.5 in a 96-wellmicrotiter plate (MTP) (Greiner, Fluotrac 200, art. nr. 655076) suitablefor fluorescence. Add 10 μl of supernatant and then add 10 μl of MUL (1mM 4-methylumbelliferyl-β-lactoside (MUL) in milliQ water) and put theMTP in the Fluostar Galaxy (BMG Labtechnologies; D-77656 Offenburg).Measure the kinetics for 16 min. (8 cycles of 120 s each) usingλ_(320 nm) (excitation) and λ_(460 nm) (emission) at 50° C. Supernatentshaving CBH activity were then subjected to Hydrophobic InteractionChromatography as described in Example 5 below.

The amino acid sequences were deduced as stated above in Example 1. Theamino acid sequences for the CBH1 homologs are shown in FIGS. 4(Hypocrea orientalis), 6 (Hypocrea schweinitzii), 8 (Trichodermakonilangbra) and 10 (Trichoderma pseudokoningii).

Thermostability of the homologs was determined as described in Example 5below.

TABLE 1 Tm measurements and comparison between the different CBH1homologous sequences. CBH1 homolog % identity Tm ΔTm Hypocrea jecorina62.5 Hypocrea schweinitzii (CBS 243.63) 96.5 61.4 −1.1 Hypocreaorientalis (PPRI 3894) 97.1 62.8 0.3 Trichoderma pseudokoningii (CBS408.91) 94.9 57.5 −5.0 Trichoderma konilangbra 93.0 59.4 −3.1

As can be seen, the CBH I cellulase homologs had a slight or negativeeffect on the thermal stability of the variant CBH I cellulases comparedto wild type. The homologs are closely related to H. jecorina CBH1; thethermal stability differences between H. jecorina and the homologs mayindicate that sites with amino acid residues different from those foundin H. jecorina CBH1 may be involved in thermostability.

Example 3 Identification of Sites Important for Stability

The amino acid sequences of the CBH1 homologs characterized in Example2, above, were aligned with the H. jecorina sequence with Vector NTIusing the Clustal W algorithm with (Nucleic Acid Research, 22 (22):4673-4680, 1994). The alignment is shown in FIG. 2.

Possible sites involved in the stability of the CBH1 enzyme weredetermined three different ways based on alignment of the sequences ofthe homologs with CBH1. In the first method, sites that differed betweenthe H. jecorina CBH1 catalytic domain and the catalytic domain of atleast one of the homologs of lower stability (i.e., excluding only H.orientalis) were identified as possible sites involved in thethermostability of CBH1. The sites identified were L6, P13, T24, Q27,S47, T59, T66, G88, N89, T160, Q186, S195, T232, E236, E239, G242, D249,N250, T281, E295, F311, E325, N327, D329, T332, A336, K354, V407, P412,T417 and/or F418 in CBH1 from Hypocrea jecorina.

In the second method, sites where the residue in H. jecoina OR H.orientalis is the same as that found in all of the decreased stabilityenzyme homologs resulted in the identification of sites that lackedcorrelation with Tm. The sites identified as retaining relevance withstability were L6, T24, Q27, S47, T59, T66, T160, Q186, S195, T232,E236, G242, D249, T281, E295, E325, N327, D329, T332, K354, and/or P412in CBH1 from Hypocrea jecorina.

In the final method, sites where H. jecorina AND H. orientalis are thesame, with the corresponding residue in H. schweinitzii being either thesame or different as in either of these two, but a different amino acidin the corresponding site of either T. konilangbra or T. pseudokoningiiwere considered as possible sites involved in thermostability of theenzyme. These sites, which empirically showed the best correlation withTm stability, were identified as Q186, S195, E325, T332 and P412.

Identification of the sites with amino acid residues different fromthose found in H. jecorina CBH1 were therefore subjected to sitesaturated mutagenesis.

Example 4 Expression of CBH1 Variants

The PCR fragments were obtained using the primers and protocolsdescribed in Example 1. The fragments were purified from an agarose gelusing the Qiagen Gel extraction KIT. The purified fragments were used toperform a clonase reaction with the pDONR™201 vector from Invitrogen®using the Gateway™ Technology instruction manual (version C) fromInvitrogen®, hereby incorporated by reference herein. Genes were thentransferred from this ENTRY vector to the destination vector (pRAXdes2)to obtain the expression vector pRAXCBH1.

Cells were transformed with an expression vector comprising a desiredcellulase encoding nucleic acid. The host cells, A. niger, were thengrown under conditions permitting expression of the desired cellulase asdescribed in Example 2.

The sites different to H. jecorina CBH1, as identified in Example 3, maybe involved in the thermostability of the variants and were thereforesubjected to site saturated mutagenesis.

Example 5 Thermostability of CBH1 Variants

CBH I cellulase variants are cloned and expressed as above (see Example4). Cel7A wild type and variants are then purified from cell-freesupernatants of these cultures by column chromatography. Proteins arepurified using hydrophobic interaction chromatography (HIC). Columnswere run on a BioCAD® Sprint Perfusion Chromatography System usingPoros® 20 HP2 resin both made by Applied Biosystems.

HIC columns are equilibrated with 5 column volumes of 0.020 M sodiumphosphate, 0.5 M ammonium sulfate at pH 6.8. Ammonium sulfate is addedto the supernatants to a final concentration of approximately 0.5 M andthe pH is adjusted to 6.8. After filtration, the supernatant is loadedonto the column. After loading, the column is washed with 10 columnvolumes of equilibration buffer and then eluted with a 10 column volumegradient from 0.5 M ammonium sulfate to zero ammonium sulfate in 0.02 Msodium phosphate pH 6.8. Cel7A is eluted approximately mid-gradientFractions are collected and pooled on the basis of reduced, SDS-PAGE gelanalysis.

The melting points are determined according to the methods of Luo, etal., Biochemistry 34:10669 and Gloss, et al., Biochemistry 36:5612.

Data is collected on the Aviv 215 circular dichroism spectrophotometer.Spectra of the variants between 210 and 260 nanometers are taken at 25°C. Buffer conditions are 50 mM Bis Tris Propane/50 mM ammoniumacetate/glacial acetic acid at pH 5.5. The protein concentration is keptbetween 0.25 and 0.5 mgs/mL. After determining the optimal wavelength tomonitor unfolding, the samples are thermally denatured by ramping thetemperature from 25° C. to 75° C. under the same buffer conditions. Datais collected for 5 seconds every 2 degrees. Partially reversibleunfolding is monitored at 230 nanometers in an 0.1 centimeter pathlength cell.

The mutations introduced into the CBH I cellulase variants have apositive effect on the thermal stability of the variant CBH I cellulasescompared to wild type.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A cell culture composition comprising a CBH1 cellulase polypeptide,wherein said polypeptide comprises a substitution or deletion at aposition corresponding to one or more of residues L6, P13, T24, Q27,S47, T59, T66, G88, T160, Q186, 5195, T232, E236, E239, G242, N250,T281, F311, N327, D329, A336, K354, V407, P412, T417 or F418 of SEQ IDNO:2 and wherein the full length of said polypeptide has at least 97%sequence identity to SEQ ID NO:2.
 2. The cell culture compositionaccording to claim 1, wherein said polypeptide comprises a substitutionselected from the group consisting of Q186(E), S195(A/F), E239S,G242(H/Y/N/S/T/D/A) and P412(T/S/A).
 3. The cell culture compositionaccording to claim 1, wherein said cells in said culture are filamentousfungus cells.
 4. The cell culture composition according to claim 3,wherein said filamentous fungus cells are selected from the groupconsisting of Trichoderma reesei, Trichoderma longibrachiatum,Trichoderma viride, Trichoderma koningii, Trichoderma harzianum,Penicillium, Humicola, Humicola insolens, Humicola grisea,Chrysosporium, Chrysosporium lucknowense, Gliocladium, Aspergillus,Fusarium, Neurospora, Hypocrea, Emericella, Aspergillus niger,Aspergillus awamori, Aspergillus aculeatus, and Aspergillus nidulans.